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Proteases are often used as biomarkers of many pathologies as well as of microbial contamination and infection. Therefore, extensive efforts are devoted to the development of protease sensors. Some applications would benefit from wireless monitoring of proteolytic activity at minimal cost, e.g., sensors embedded in care products like wound dressings and diapers to track wound and urinary infections. Passive (batteryless) and chipless transponders stand out among wireless sensing technologies when low cost is a requirement. Here, we developed and extensively characterized a composite material that is biodegradable but still highly stable in aqueous media, whose proteolytic degradation could be used in these wireless transponders as a transduction mechanism of proteolytic activity. This composite material consisted of a cross-linked gelatin network with incorporated caprylic acid. The digestion of the composite when exposed to proteases results in a change of its resistivity, a quantity that can be wirelessly monitored by coupling the composite to an inductor-capacitor resonator, i.e., an antenna. We experimentally proved this wireless sensor concept by monitoring the presence of a variety of proteases in aqueous media. Moreover, we also showed that detection time follows a relationship with protease concentration, which enables quantification possibilities for practical applications.

The Mo-dependent enzyme YiiM enzyme from Escherichia coli is a member of the sulfite oxidase family and shares many similarities with the well-studied human mitochondrial amidoxime reducing component (mARC). We have investigated YiiM catalysis using electrochemical and spectroscopic methods. EPR monitored redox potentiometry found the active site redox potentials to be Mo

The porous carbon-based materials (PCMs) have recently been attracted to many applications, specifically energy conversion and storage devices, due to their excellent electrocatalytic activities and abundant nature. The PCMs possess fascinating properties, including unique porosity, low cost, higher conductivity, fast mass transport for easy access of analytes and durability. Here, we have highlighted the different forms of PCMs, such as nanoporous, mesoporous, graphene, metal, and metal oxides based carbon composites for water-splitting reactions and their significance in important energy storage devices (metal-air batteries and fuel cells). This review is mainly focused on the various approaches for the preparation of porous carbon-based materials over the last ten years and the fabricated materials that could be applied in large-scale practical energy-related applications. In addition, we summarize the most promising strategies for the development of morphological structural based electrocatalysts to enhance their electrocatalytic performances for both OER and HER activities are discussed.

Continuous and automated bacteria detection is pivotal for a myriad of biomedical, food safety and envi-ronmental applications. This work presents the fabrication of a prototype of a passive (battery-free) radio frequency sensor for wireless detection of bacteria. The sensing mechanism is based on the bacterial-induced (proteases and peptidases) degradation of glutaraldehyde (GTA) cross-linked gelatin-caprylic acid (CA) composite film. Proteolytic degradation of the film resulted in a decrease of its resistivity, a quan-tity that could be wirelessly monitored by coupling the film to a radio-frequency antenna (an inductor-capacitor resonator) and monitoring the frequency for which the transferred power between this antenna and another antenna connected to a Vector Network Analyzer (VNA) was maximized. We experimen-tally proved this concept by monitoring E.coli bacteria in aqueous medium and detected at 18.0 +/- 2.8 h, 23.5 +/- 0.7 h, 27.0 +/- 2.8 h, 40.5 +/- 3.5 h, 45.5 +/- 0.7 h for the initial E.coli concentration of 3.2 +/- 10(8) , 6.8 +/- 10(7) , 2.3 +/- 10(6) , 4.3 +/- 10(5) , and 3.6 +/- 10(4) CFU/mL, respectively. Further, the E.coli induced degrada-tion of the composite film was investigated by evaluating the thickness of the film by optical microscopy as well as morphology by scanning electron microscopy techniques. (C) 2021 Published by Elsevier Ltd.

The global market of disposable biosensors has experienced tremendous growth in recent years because of the increasing demand for testing outside of central laboratories. These types of sensors provide critical analytical information for anyone, anywhere and at any time, without contamination and recalibration. Several materials have been incorporated in low-cost electrodes for the development of disposable electrochemical sensors for biomedical applications. Among them, conducting polymers (CPs) have proven that they are the most suitable candidate due to their excellent properties. These properties include high conductivity, redox behaviours, inkjet printing ability, active functionalities in the polymer backbone, biocompatibility, cost-effectiveness and mass production, etc. CPs containing functional groups such as NH2 and COOH have been used for the covalent and electrostatic attachment of several biocatalysts. Notably, the redox-active characteristics of CPs offer to enable the biosensors to operate at very low potential without the use of external redox reagents. Further, CPs have displayed a high selectivity for analytes while fabricating by molecularly imprinting technique. Moreover, the biosensing properties of CPs have been enhanced by the integration of nanomaterials. This chapter focusses on recent developments of CPs-based disposable electrochemical biosensors for biomedical applications.

We report electrochemically driven catalysis of the complex molybdoenzyme steroid C25 dehydrogenase (S25DH) from the β-Proteobacterium Sterolibacterium denitrificans. S25DH catalyzes the oxygen-independent regioselective hydroxylation of the tertiary C25 atom of sterols and also their derivatives. Cholest-4-en-3-one is a native substrate for S25DH, which produces 25-hydroxy-cholest-4-en-3-one as a product of catalytic turnover. Cholecalciferol (vitD3) is also a substrate. S25DH was immobilized on a modified Au working electrode with the co-adsorbent chitosan. The complexes ferricyanide ([Fe(CN)6]3-) and ferrocenium methanol (FM+) are effective artificial electron acceptors from S25DH and act as mediators of electron transfer between the electrode and the enzyme. 2-Hydroxypropyl-β-cyclodextrin (HPCD) was employed as a sterol solubilizer in addition to 2-methoxyethanol. The catalytic activity varied depending upon the concentration of solubilizer in the reaction mixture. Parallel studies using ferricyanide as a chemical (as opposed to electrochemical) oxidant coupled to HPLC analysis show that S25DH is capable of oxidising both vitD3 and its less stable isomer pre-vitD3 and that the former substrate is stabilised by HPCD.

Metal-air batteries and fuel cells are considered the most promising highly efficient energy storage systems because they possess long life cycles, high carbon monoxide (CO) tolerance, and low fuel crossover ability. The use of energy storage technology in the transport segment holds great promise for producing green and clean energy with lesser greenhouse gas (GHG) emissions. In recent years, nanoscale based electrocatalysts have shown remarkable electrocatalytic performance towards the construction of sustainable energy-related devices/applications, including fuel cells, metal-air battery and water-splitting processes. This review summarises the recent advancement in the development of nanoscale-based electrocatalysts and their energy-related electrocatalytic applications. Further, we focus on different synthetic approaches employed to fabricate the nanomaterial catalysts and also their size, shape and morphological related electrocatalytic performances. Following this, we discuss the catalytic reaction mechanism of the electrochemical energy generation process, which provides close insight to develop a more efficient catalyst. Moreover, we outline the future perspectives and challenges pertaining to the development of highly efficient nanoscale-based electrocatalysts for green energy storage technology.

A central conserved arginine, first identified as a clinical mutation leading to sulfite oxidase deficiency, is essential for catalytic competency of sulfite oxidizing molybdoenzymes, but the molecular basis for its effects on turnover and substrate affinity have not been fully elucidated. We have used a bacterial sulfite dehydrogenase, SorT, which lacks an internal heme group, but transfers electrons to an external, electron accepting cytochrome, SorU, to investigate the molecular functions of this arginine residue (Arg78). Assay of the SorT Mo centre catalytic competency in the absence of SorU showed that substitutions in the central arginine (R78Q, R78K and R78M mutations) only moderately altered SorT catalytic properties, except for R78M which caused significant reduction in SorT activity. The substitutions also altered the Mo-centre redox potentials (MoVI/V potential lowered by ca. 60-80mV). However, all Arg78 mutations significantly impaired the ability of SorT to transfer electrons to SorU, where activities were reduced 17 to 46-fold compared to SorTWT, precluding determination of kinetic parameters. This was accompanied by the observation of conformational changes in both the introduced Gln and Lys residues in the crystal structure of the enzymes. Taking into account data collected by others on related SOE mutations we propose that the formation and maintenance of an electron transfer complex between the Mo centre and electron accepting heme groups is the main function of the central arginine, and that the reduced turnover and increases in KMsulfite are caused by the inefficient operation of the oxidative half reaction of the catalytic cycle in enzymes carrying these mutations.

Presently, the global energy demand for increasing clean and green energy consumption lies in the development of low-cost, sustainable, economically viable and eco-friendly natured electrochemical conversion process, which is a significant advancement in different morphological types of advanced electrocatalysts to promote their electrocatalytic properties. Herein, we overviewed the recent advancements in oxygen evolution reactions (OERs), including easy electrode fabrication and significant action in water-splitting devices. To date, various synthetic approaches and modern characterization techniques have effectively been anticipated for upgraded OER activity. Moreover, the discussed electrode catalysts have emerged as the most hopeful constituents and received massive appreciation in OER with low overpotential and long-term cyclic stability. This review article broadly confers the recent progress research in OER, the general mechanistic approaches, challenges to enhance the catalytic performances and future directions for the scientific community.

We report an electrochemical nitrate biosensor incorporating the enzyme nitrate reductase (NR) from the fungus Neurospora crassa entrapped within a polymeric chitosan matrix on a glassy carbon (GC) electrode. The artificial electron mediator anthraquinone sulfonate (AQ) is utilized as an electron transfer partner (co-substrate) for NR. The NR enzyme was effectively immobilized within the chitosan matrix without the need for a dialysis membrane, and exhibited high sensitivity and selectivity for nitrate using either rotating disk cyclic voltammetry or constant potential amperometry methods. A particular feature was the longevity of the biosensor which retained more than 70% activity over a period of 3 months. The practical application of the biosensor was demonstrated by the determination of nitrate concentrations in lake and river water samples without any pre-treatment, and the results were validated with a reliable spectroscopic assay.

As recently as 2006 the mitochondrial amidoxime reducing component (mARC) was identified as the fourth and last Mo enzyme present in humans. Its physiological role remains unknown. mARC is capable of reducing a variety of N-hydroxylated compounds such as amidoximes to their corresponding amidine and there is considerable interest in this enzyme from a pharmaceutical perspective. mARC is a target for N-hydroxylated pro-drugs that may be reductively activated intracellularly to release potent drugs such as cationic amidinium ions, which exhibit a broad spectrum of activity as antithrombotics and against various bacteria and parasites. In the quest for a rapid screen of new mARC substrates and inhibitors we present an electrochemical method which utilizes the natural electron partner of mARC, cytochrome b5, coupled to an electrochemical electrode. Mediated electron transfer from the electrode via cytochrome b5 to mARC results in a catalytic current in the presence of substrate. Keywords: mARC, Molybdenum, Enzyme, Voltammetry, Cytochrome

Direct electrochemistry of human sulfite oxidase (HSO) has been achieved on carboxylate-terminated self-assembled monolayers cast on a Au working electrode in the presence of the promoter chitosan. The modified electrode facilitates a well-defined nonturnover redox response from the heme cofactor (FeIII/II) in 750 mM Tris, MOPS, and bicine buffer solutions. The formal redox potential of the nonturnover response varies slightly depending on the nature of the thiol monolayer on the Au electrode. Upon addition of sulfite to the cell a pronounced catalytic current from HSO-facilitated sulfite oxidation is observed. The measured catalytic rate constant (kcat) is around 0.2 s–1 (compared with 26 s–1 obtained from solution assays), which indicates that interaction of the enzyme with the electrode lowers overall catalysis although native behavior is retained in terms of substrate concentration dependence, pH dependence, and inhibition effects. In contrast, no catalytic activity is observed when HSO is confined to...

We demonstrate electrochemically driven catalytic voltammetry of the Mo-dependent sulfite dehydrogenase (SorT) from the -Proteobacterium Sinorhizobium meliloti with its physiological electron acceptor, the c-type cytochrome (SorU) with both proteins co-adsorbed on a chemically modified Au working electrode. Both SorT and SorU were constrained under a perm-selective dialysis membrane with the biopolymer chitosan as a co-adsorbate while the electrode was modified by a 3-mercaptopropionate self-assembled monolayer cast on the Au electrode. Cyclic voltammetry of the SorU protein reveals a well-defined quasi-reversible FeIII/II redox couple at +130 mV vs NHE in 100 mM phosphate buffer solution (pH 7.0). Introduction of wild type sulfite dehydrogenase (SorTWT) and sulfite transforms this transient SorU voltammetric response into a sigmoidal catalytic wave which increases with sulfite concentration before eventually saturating. In addition to the wild type enzyme, the variants SorTR78K, SorTR78M and SorTR78Q were also examined electrochemically in an effort to better understand the role of amino acid residue Arg78 which is in the vicinity of the Mo active site of SorT.

CO dehydrogenase (CODH) from the Gram-negative bacterium Oligotropha carboxidovorans is a complex metalloenzyme from the xanthine oxidase family of molybdenum-containing enzymes, bearing a unique binuclear Mo-S-Cu active site in addition to two [2Fe-2S] clusters (FeSI and FeSII) and one equivalent of FAD. CODH catalyzes the oxidation of CO to CO2 with the concomitant introduction of reducing equivalents into the quinone pool, thus enabling the organism to utilize CO as sole source of both carbon and energy. Using a variety of EPR monitored redox titrations and spectroelectrochemistry, we report the redox potentials of CO dehydrogenase at pH 7.2 namely MoVI/V, MoV/IV, FeSI2+/+, FeSII2+/+, FAD/FADH and FADH/FADH−. These potentials are systematically higher than the corresponding potentials seen for other members of the xanthine oxidase family of Mo enzymes, and are in line with CODH utilising the higher potential quinone pool as an electron acceptor instead of pyridine nucleotides. CODH is also active when immobilised on a modified Au working electrode as demonstrated by cyclic voltammetry in the presence of CO.

High efficient, low-cost and environmentally friendly-natured bi-functional-based perovskite electrode catalysts (BFPEC) are receiving increasing attention for oxygen reduction/oxygen evolution reaction (ORR/OER), playing an important role in the electrochemical energy conversion process using fuel cells and rechargeable batteries. Herein, we highlighted the different kinds of synthesis routes, morphological studies and electrode catalysts with A-site and B-site substitution co-substitution, generating oxygen vacancies studies for boosting ORR and OER activities. However, perovskite is a novel type of oxide family, which shows the state-of-art electrocatalytic performances in energy storage device applications. In this review article, we go through different types of BFPECs that have received massive appreciation and various strategies to promote their electrocatalytic activities (ORR/OER). Based on these various properties and their applications of BFPEC for ORR/OER, the general mechanism, catalytic performance and future outlook of these electrode catalysts have also been discussed.

Nitrate reductase (NR) from the fungus Neurospora crassa is a complex homodimeric metallo-flavoenzyme, where each protomer contains three distinct domains; the catalytically active terminal molybdopterin cofactor, a central heme-containing domain, and an FAD domain which binds with the natural electron donor NADPH. Here, we demonstrate the catalytic voltammetry of variants of N. crassa NRs on a modified Au electrode with the electrochemically reduced forms of benzyl viologen (BV2+) and anthraquinone sulfonate (AQS−) acting as artificial electron donors. The biopolymer chitosan used to entrap NR on the electrode non-covalently and the enzyme film was both stable and highly active. Electrochemistry was conducted on two distinct forms; one lacking the FAD cofactor and the other lacking both the FAD and heme cofactors. While both enzymes showed catalytic nitrate reductase activity, removal of the heme cofactor resulted in a more significant effect on the rate of nitrate reduction. Electrochemical simulation was carried out to enable kinetic characterisation of both the NR:nitrate and NR:mediator reactions.

Herein, we report the electrocatalytic voltammetry of holo and heme-free human sulfite oxidase (HSO) mediated by the synthetic iron complexes 1,2-bis(1,4,7-triaza-1-cyclononyl)ethane iron(III) bromide, ([Fe(dtne)]Br.3HO), potassium ferricyanide (K[Fe(CN)]), and ferrocene methanol (FM) at a 5-(4′-pyridinyl)-1,3,4-oxadiazole-2-thiol (Hpyt) modified gold working electrode. Holo HSO contains two electroactive redox cofactors, comprising a mostly negatively charged cyt b (heme) domain and a Mo cofactor (Moco) domain (the site of sulfite oxidation), where the surface near the active site is positively charged. We anticipated different catalytic voltammetry based on either repulsive or attractive electrostatic interactions between the holo or heme-free enzymes and the positively or negatively charged redox mediators. Both holo and heme-free HSO experimental catalytic voltammetry has been modeled by using electrochemical simulation across a range of sweep rates and concentrations of substrate and both positive and negatively charged electron acceptors ([Fe(dtne)], [Fe(CN)] and FM), which provides new insights into the kinetics of the HSO catalytic mechanism. These mediator complexes have almost the same redox potential (all lying in the range +415 to +430 mV vs. NHE) and, thus, deliver the same driving force for electron transfer with the Mo cofactor. However, differences in the electrostatic affinities between HSO and the mediator have a significant influence on the electrocatalytic response.

We report the first direct (unmediated) catalytic electrochemistry of a eukaryotic nitrate reductase (NR). NR from the filamentous fungus Neurospora crassa, is a member of the mononuclear molybdenum enzyme family and contains a Mo, heme and FAD cofactor which are involved in electron transfer from NAD(P)H to the (Mo) active site where reduction of nitrate to nitrite takes place. NR was adsorbed on an edge plane pyrolytic graphite (EPG) working electrode. Non-turnover redox responses were observed in the absence of nitrate from holo NR and three variants lacking the FAD, heme or Mo cofactor. The FAD response is due to dissociated cofactor in all cases. In the presence of nitrate, NR shows a pronounced cathodic catalytic wave with an apparent Michaelis constant (KM) of 39μM (pH7). The catalytic cathodic current increases with temperature from 5 to 35°C and an activation enthalpy of 26kJmol(-1) was determined. In spite of dissociation of the FAD cofactor, catalytically activity is maintained.

Mediated electrocatalytic voltammetry of human sulfite oxidase (HSO) is demonstrated with synthetic one electron transfer iron complexes bis(1,4,7-triazacyclononane)iron(III) ([Fe(tacn) 2 ] 3+ ) and 1,2-bis(1,4,7-triaza-1-cyclononyl)ethane iron(III) ([Fe(dtne)] 3+ ) at a glassy carbon working electrode. The two synthetic electron acceptors for HSO, differing in redox potential by 270 mV, deliver different driving forces for electrocatalysis. Digital simulation of the catalytic voltammetry was achieved with single set of enzyme-dependent kinetic parameters that reproduced the experimental data across a range of sweep rates, and sulfite and mediator concentrations. Amperometry carried out in a stirred solution with the lower potential mediator [Fe(tacn) 2 ] 3+ was optimised and exhibited a linear increase in steady state current in the sulfite concentration range 5.0 × 10 −6 to 8.0 × 10 −4 M with a detection limit of 0.2 pM (S/N = 3). The HSO coupled electrode was successfully used for the determination of sulfite concentration in white wine and beer samples and the results validated with a standard spectrophotometric method.

MtsZ is a molybdenum-containing methionine sulfoxide reductase that supports virulence in the human respiratory pathogen Haemophilus influenzae (Hi). HiMtsZ belongs to a group of structurally and spectroscopically uncharacterized S-/N-oxide reductases, all of which are found in bacterial pathogens. Here, we have solved the crystal structure of HiMtsZ, which reveals that the HiMtsZ substrate-binding site encompasses a previously unrecognized part that accommodates the methionine sulfoxide side chain via interaction with His182 and Arg166. Charge and amino acid composition of this side chain-binding region vary and, as indicated by electrochemical, kinetic, and docking studies, could explain the diverse substrate specificity seen in closely related enzymes of this type. The HiMtsZ Mo active site has an underlying structural flexibility, where dissociation of the central Ser187 ligand affected catalysis at low pH. Unexpectedly, the two main HiMtsZ electron paramagnetic resonance (EPR) species resembled not only a related dimethyl sulfoxide reductase but also a structurally unrelated nitrate reductase that possesses an Asp-Mo ligand. This suggests that contrary to current views, the geometry of the Mo center and its primary ligands, rather than the specific amino acid environment, is the main determinant of the EPR properties of mononuclear Mo enzymes. The flexibility in the electronic structure of the Mo centers is also apparent in two of three HiMtsZ EPR-active Mo(V) species being catalytically incompetent off-pathway forms that could not be fully oxidized.

We report the first electrochemical study of a lanthanoid-dependent methanol dehydrogenase (Eu-MDH) from the acidophilic verrucomicrobial methanotroph Methylacidiphilum fumariolicum SolV with its own physiological cytochrome cGJ electron acceptor. Eu-MDH harbours a redox active 2,7,9-tricarboxypyrroloquinoline quinone (PQQ) cofactor which is non-covalently bound but coordinates trivalent lanthanoid elements including Eu3+ . Eu-MDH and the cytochrome were co-adsorbed with the biopolymer chitosan and cast onto a mercaptoundecanol (MU) monolayer modified Au working electrode. Cyclic voltammetry of cytochrome cGJ reveals a well-defined quasi-reversible FeIII/II redox couple at +255 mV vs. NHE at pH 7.5 and this response is pH independent. The reversible one-electron response of the cytochrome cGJ transforms into a sigmoidal catalytic wave in the presence of Eu-MDH and its substrates (methanol or formaldehyde). The catalytic current was pH-dependent and pH 7.3 was found to be optimal. Kinetic parameters (pH dependence, activation energy) obtained by electrochemistry show the same trends as those obtained from an artificial phenazine ethosulfate/dichlorophenol indophenol assay.

Creating well-ordered nanoporosity in biomolecules promises stability and activity, offering access to an even wider range of application possibilities. Here, the preparation of nanoporous protein films containing cytochrome c protein molecules is reported through a soft-templating strategy using polystyrene (PS) spheres of different sizes as templates. The stability of the cytochrome c film is demonstrated through electrochemistry studies to show a reusable nature of these films over a long period of time. The size of the PS spheres is varied to tune the pore diameter and the thickness of the cytochrome c films, which are quite stable and highly selective for sensing toxic acidic vapors. The fusion of the templating strategy and the self-assembly of biomolecules may offer various possibilities by generating a new series of porous biomolecules including enzymes with different molecular weights and diameters, peptides, antibodies, and DNA with interesting catalytic, adsorption, sensing, and electronic properties. Refereed/Peer-reviewed

Sulfite dehydrogenase from the soil bacterium Sinorhizobium meliloti (SorT) is a periplasmic, homodimeric molybdoenzyme with a molecular mass of 78 kDa. It differs from most other well studied sulfite oxidizing enzymes, as it bears no heme cofactor. SorT does not readily reduce ferrous horse heart cytochrome c which is the preferred electron acceptor for vertebrate sulfite oxidases. In the present study, ferrocene methanol (FM) (in its oxidized ferrocenium form) was utilized as an artificial electron acceptor for the catalytic SorT sulfite oxidation reaction. Cyclic voltammetry of FM was used to generate the active form of the mediator at the electrode surface. The FM-mediated catalytic sulfite oxidation by SorT was investigated by two different voltammetric methods, namely, (i) SorT freely diffusing in solution and (ii) SorT confined to a thin layer at the electrode surface by a semipermeable dialysis membrane. A single set of rate and equilibrium constants was used to simulate the catalytic voltammograms performed under different sweep rates and with various concentrations of sulfite and FM which provides new insights into the kinetics of the SorT catalytic mechanism. Further, we were able to model the role of the dialysis membrane in the kinetics of the overall catalytic system.

The tachykinin neuropeptide family, which includes substance P and neurokinin B, is involved in a wide array of biological functions. Among these is the ability to protect against the neurotoxic processes in Alzheimer's Disease, but the mechanisms driving neuroprotection remain unclear. Dysregulation of metal ions, particularly copper, iron and zinc is a common feature of Alzheimer's Disease, and other amyloidogenic disorders. Copper is known to be released from neurons and recent work has shown that some tachykinins can bind Cu(II) ions, and that neurokinin B can inhibit copper uptake into astrocytes. We have now examined whether neurokinin B is capable of binding Cu(I), which is predicted to be available in the synapse. Using a combination of spectroscopic techniques including cyclic voltammetry and magnetic resonance we show that neurokinin B can bind Cu(I) either directly from added CuCl or by reduction of Cu(II)-bound neurokinin B. The results showed that the Cu(I) binding site differs greatly to that of Cu(II) and involves thioether coordination via Met2 and Met10 and an imidazole nitrogen ligand from His3. The Cu(I) coordination is also different to the site adopted by Ag(I). During changes in oxidation state, copper remains bound to neurokinin B despite large changes to the inner coordination sphere. We predict that neurokinin B may be involved in synaptic copper homeostasis.

This paper describes a highly sensitive electrochemical (voltammetric) determination of sulfite using a combination of Starkeya novella sulfite dehydrogenase (SDH), horse heart cytochrome c (cyt c), and a self-assembled monolayer of 11-mercaptoundecanol (MU) cast on a gold electrode. The biosensor was optimized in terms of pH and the ratio of cyt c/SDH. The electrocatalytic oxidation current of sulfite increased linearly from 1 to 6 microM at the enzyme-modified electrode with a correlation coefficient of 0.9995 and an apparent Michaelis constant (K(M,app)) of 43 microM. Using an amperometric method, the low detection limit for sulfite at the enzyme-modified electrode was 44 pM (signal-to-noise ratio = 3). The modified electrode retained a stable response for 3 days while losing only ca. 4% of its initial sensitivity during a 2 week storage period in 50 mM Tris buffer solution at 4 degrees C. The enzyme electrode was successfully used for the determination of sulfite in beer and white wine samples. The results of these electrochemical analyses agreed well with an independent spectrophotometric method using Ellman's reagent, but the detection limit was far superior using the electrochemical method.

The bacterial xanthine dehydrogenase (XDH) from Rhodobacter capsulatus was immobilized on an edge-plane pyrolytic graphite (EPG) electrode to construct a hypoxanthine/xanthine biosensor that functions at physiological pH. Phenazine methosulfate (PMS) was used as a mediator which acts as an artificial electron-transfer partner for XDH. The enzyme catalyzes the oxidation of hypoxanthine to xanthine and also xanthine to uric acid by an oxidative hydroxylation mechanism. The present electrochemical biosensor was optimized in terms of applied potential and pH. The electrocatalytic oxidation response showed a linear dependence on the xanthine concentration ranging from 1.0 × 10(-5) to 1.8 × 10(-3) M with a correlation coefficient of 0.994. The modified electrode shows a very low detection limit for xanthine of 0.25 nM (signal-to-noise ratio = 3) using controlled potential amperometry.

We report the mediated electrocatalytic voltammetry of the molybdoenzyme xanthine dehydrogenase (XDH) from Rhodobacter capsulatus at a thiol-modified Au electrode. The 2-electron acceptor N-methylphenazinium methanesulfonate (phenazine methosulfate, PMS) is an effective artificial electron transfer partner for XDH instead of its native electron acceptor NAD(+). XDH catalyzes the oxidative hydroxylation of hypoxanthine to xanthine and xanthine to uric acid. Cyclic voltammetry was used to generate the active (oxidized) form of the mediator. Simulation of the catalytic voltammetry across a broad range of substrate and PMS concentrations at different sweep rates was achieved with the program DigiSim to yield a set of consistent rate and equilibrium constants that describe the catalytic system. This provides the first example of the mediated electrochemistry of a xanthine dehydrogenase (or oxidase) that is uncomplicated by interference from product oxidation. A remarkable two-step, sequential oxidation of hypoxanthine to uric acid via xanthine by XDH is observed.

We report here the highly sensitive and selective electrochemical determination of 3,4-dihydroxyphenylacetic acid (DOPAC), one of the dopamine metabolites in the presence of important interferents ascorbic acid (AA) and uric acid (UA) using an ultrathin electropolymerized film of 5-amino-1,3,4-thiadiazole-2-thiol (p-ATT) modified glassy carbon (GC) electrode in 0.20M phosphate buffer solution (pH5.0). The bare GC electrode fails to resolve the oxidation peaks of AA, DOPAC and UA in a mixture. Further, the oxidation peak potentials of them were shifted to more positive potential with decreased peak currents in the subsequent cycles. On the other hand, the p-ATT modified electrode not only separated the voltammetric signals of AA, DOPAC and UA but also enhanced their peak currents. The amperometric current response was increased linearly with increasing DOPAC concentration in the range of 4.0×10 − 8 to 1.0×10 − 5 M and the detection limit was found to be 150pM (S/N = 3).

The enzyme xanthine dehydrogenase (XDH) from the purple photosynthetic bacterium Rhodobacter capsulatus catalyzes the oxidation of hypoxanthine to xanthine and xanthine to uric acid as part of purine metabolism. The native electron acceptor is NAD(+) but herein we show that uric acid in its 2-electron oxidized form is able to act as an artificial electron acceptor from XDH in an electrochemically driven catalytic system. Hypoxanthine oxidation is also observed with the novel production of uric acid in a series of two consecutive 2-electron oxidation reactions via xanthine. XDH exhibits native activity in terms of its pH optimum and inhibition by allopurinol.

This paper reports the selective determination of norepinephrine (NEP) in the presence of very important interferences, ascorbic acid (AA) and uric acid (UA) using electropolymerized film of 2-amino-1,3,4-thiadiazole (p-ATD) modified glassy carbon (GC) electrode in 0.20 M phosphate buffer solution (pH 5.0). The bare GC electrode does not separate the voltammetric signals of AA, NEP and UA. However, p-ATD modified GC electrode not only resolved the voltammetric signals of AA, NEP and UA with potential differences of 150 and 130 mV between AA–NEP and NEP–UA, respectively but also dramatically enhanced the oxidation peak currents of them when compared to bare GC electrode. The modified electrode showed an excellent selectivity towards NEP even in the presence of 100-fold excess of AA and UA. The amperometric current was linearly increased from 40 nM to 25 μM for NEP and the lowest detection limit was found to be 0.17 nM (S/N = 3). The practical application of the modified electrode was demonstrated by determining NEP in norepinephrine hydrochloride injection.

We report the selective determination of homocysteine (HCY) in the presence of one of the very important interferents, ascorbic acid (AA) using electropolymerized film of 2-amino-1,3,4-thiadiazole (ATD) modified glassy carbon electrode (GCE) at physiological pH for the first time. An atomic force microscopic image showed that the electropolymerized film of ATD (p-ATD) formed a spherical like structure with a thickness of 25 nm. This nanostructured film oxidized HCY at 0.55 V while bare GCE failed to oxidize it at physiological pH. Further, p-ATD modified electrode successfully separated the voltammetric signals of AA and HCY with a peak separation of 490 mV. The amperometric current was increased linearly from 100 to 1400 nM HCY and achieved the detection limit of 51 pM (S/N = 3). The present modified electrode showed better recoveries for spiked HCY in human blood serum samples.

Mercaptothiadiazole ligands functionalized with thiol (2,5-dimercapto-1,3,4-thiadiazole (DMT)) and methyl (5-methyl-2-mercapto-1,3,4-thiadiazole (MMT)) groups capped onto citrate capped gold nanoparticles (C-AuNPs) by ligand exchange reaction was investigated by UV–vis spectroscopy, FT-IR spectroscopy and transmission electron microscopy (TEM) techniques. The surface plasmon resonance band at 522 nm for C-AuNPs was shifted to 530 nm after capping with DMT whereas an additional band was observed at 630 nm due to aggregation in addition to a shift in the band at 522 nm after capping of MMT onto C-AuNPs. Thus, capping of DMT onto C-AuNPs leads to the formation of stable AuNPs while capping of MMT leads to the formation of unstable AuNPs. FT-IR studies show that the citrate ions were completely replaced by both DMT and MMT ligands from the AuNPs. TEM images indicate that the size and shape of the AuNPs remain same after capping of these ligands.

This paper describes the simultaneous determination of ascorbic acid (AA), dopamine (DA), uric acid (UA) and xanthine (XN) using an ultrathin electropolymerized film of 2-amino-1,3,4-thiadiazole (p-ATD) modified glassy carbon (GC) electrode in 0.20 M phosphate buffer solution (pH 5.0). Bare GC electrode failed to resolve the voltammetric signals of AA, DA, UA and XN in a mixture. On the other hand, the p-ATD modified electrode separated the voltammetric signals of AA, DA, UA and XN with potential differences of 110, 152 and 392 mV between AA-DA, DA-UA and UA-XN, respectively and also enhanced their oxidation peak currents. The modified electrode could sense 5 microM DA and 10 microM each UA and XN even in the presence of 200 microM AA. The oxidation currents were increased from 30 to 300 microM for AA, 5 to 50 microM for DA and 10 to 100 microM for each UA and XN, and the lowest detection limit was found to be 2.01, 0.33, 0.19 and 0.59 microM for AA, DA, UA and XN, respectively (S/N=3). The practical application of the present modified electrode was demonstrated by the determination of AA, UA and XN in human urine samples.

This paper describes the selective electrochemical determination of paracetamol (PA) in the presence of important interferent, ascorbic acid (AA) using an ultrathin electropolymerized film of 5-amino-1,3,4-thiadiazole-2-thiol (p-ATT) modified glassy carbon (GC) electrode in 0.20 M phosphate buffer solution (pH 7.20). Bare GC electrode failed to resolve the voltammetric signals of AA and PA in a mixture. On the other hand, the p-ATT modified electrode not only separated the voltammetric signals of AA and PA but also enhanced their peak currents. We achieved the lowest detection lit-nit of 0.34 nM (S/N=3) for PA at p-ATT modified electrode.

We report the electropolymerization of 2-amino-1,3,4-thiadiazole (ATD) on glassy carbon (GC) and indium tin oxide (ITO) electrodes in 0.10 M H2SO4. The electropolymerized ATD (p-ATD) film was characterized by cyclic voltammetry, attenuated total reflectance (ATR)-FT-IR spectroscopy, X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). The AFM image showed that the p-ATD formed a spherical-like structure with a thickness of 25 nm. XPS of the p-ATD film showed binding energies at 398.7, 400.3 and 401.3 eV in the N 1s region corresponding to -N=, -NH- and -N+H-, respectively, and at 285.5 and 287.0 eV in the C 1s region corresponding to C-N and C=N, respectively. The appearance of binding energies at 285.5 and 287.0 eV confirmed that the p-ATD film proceeded via C-N and C=N linkages and not via C-C or C=C linkages. The p-ATD film deposited on the GC electrode was successfully used for the determination of ascorbic acid (AA) at physiological pH. The amperometric current was increased linearly from 7.5 x 10(-8) to 2.0 x 10(-5), and the detection limit was found to be 0.28 nM (S/N=3). (C) 2009 Elsevier Ltd. All rights reserved.

Adsorption of a heteroaromatic dithiol, 2,5-dimercapto-1,3,4-thiadiazole (DMT), on Au surface from a completely deaerated aqueous solution leads to the formation of a multilayer assembly via hydrogen bonding with water molecules, whereas adsorption from acetonitrile, ethanol, dimethyl sulfoxide, and chloroform solutions leads to the formation of a monolayer. Cyclic voltammetry (CV), attenuated total reflectance (ATR) infrared spectroscopy, Raman spectroscopy, high-resolution X-ray photoelectron spectroscopy (XPS), and scanning electron microscopy (SEM) were used to characterize the monolayer and multilayer assemblies of DMT on Au surface. Since DMT contains two S−H groups, it chemisorbs on Au surface through one of its two S−H groups, while the other S−H group is pointing away from the surface. The presence of free S−H groups on the Au surface was confirmed by CV, ATR-FT-IR, and XPS. It is presumed that the free S−H groups of DMT on the Au surface form a hydrogen bond with the water molecules. Subsequentl...

This paper reports a highly sensitive and selective determination of nitrite at physiological pH using electropolymerized film of 5-amino-1,3,4-thiadiazole-2-thiol (p-ATT) on glassy carbon electrode (GCE) by amperometry. Bare GCE oxidizes nitrite at 0.96 V in the first cycle and in the subsequent cycles the oxidation peak was shifted to 1.03 V with decreased peak current. However, p-ATT modified electrode oxidizes nitrite at 0.84 V with an enhanced peak current and this oxidation peak was very much stable in the subsequent cycles. Using amperometric method, we achieved the lowest detection of 50 nM nitrite for the first time. Further, the amperometric current increases linearly while increasing the nitrite concentration from 5.0 × 10−8 to 1.6 × 10−5 M and a detection limit was found to be 340 pM (S/N = 3). The modified electrode was used to determine nitrite in the presence of 1000-fold excess interferents such as Na+, F−, Ca2+, Cl−, Mg2+, SO42-, NH4+, K+, CO32-, NO3-, glucose, urea and oxalate. The practical application of the present method was successfully applied to the determination of nitrite in water samples. Keywords: Electropolymerization, Nitrite, Amperometry, Sensitivity, Selectivity

We report the electropolymerization of 5-amino-2-mercapto-1,3,4-thiadiazole (AMT) in 0.1 M H2SO4 on glassy carbon electrode (GCE) and utilization of the resulting polymer film for the selective determination of l-cysteine (CY) at physiological pH for the first time. The electropolymerized film was characterized by cyclic voltammetry (CV), atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS). AFM image shows a homogeneous film containing spherical structure with a thickness of ∼25 nm for p-AMT deposited by 15 cycles. The binding energies at 163.5, 400.2 and 398.8 eV in the XPS corroborate that the p-AMT was linked by S–S, HN–NH and NN groups. The p-AMT film successfully separates the voltammetric signals of CY and ascorbic acid (AA) with a potential difference of 480 mV which is higher than the previous reports. Keywords: Electropolymerization, 5-Amino-2-mercapto-1,3,4-thiadiazole, l-Cysteine, XPS, AFM

The electrocatalytic activity of different sizes (2.6, 12.6, 20, 40 and 60 nm) of gold nanoparticles (AuNPs) immobilized onto (3-mercaptopropyl)-trimethoxysilane sol–gel modified Au electrodes (Au-MPTS) were examined by taking biologically important compounds such as ascorbic acid (AA), uric acid (UA) and 3,4-dihydroxyphenylacetic acid (DOPAC). The oxidation current of these compounds decreases and the oxidation potentials of them were shifted to more positive potential when the size of the immobilized AuNPs increases. Since AuNPs with smaller size provides higher surface area when compared to AuNPs with larger size, higher oxidation currents were observed for these compounds at smaller size AuNPs modified electrodes. AFM images show that AuNPs with 20 nm size were densely packed onto MPTS sol–gel film and generated continuous array whereas AuNPs with 60 nm size were less densely packed. The diffuse reflectance spectra (DRS) of 2.6 nm and 60 nm AuNPs immobilized onto MPTS sol–gel film show the absorption maximum around 520 nm and 540 nm, respectively indicating that the immobilized AuNPs were not aggregated in the sol–gel network. The AuNPs immobilized Au-MPTS electrodes successfully resolve the oxidation peaks of AA and UA in 0.2 M phosphate buffer solution (pH 7.2) whereas bare Au electrode fails to resolve the oxidation peaks of them. Interestingly the peak separation between AA and UA was identical (180 mV) irrespective of the size of AuNPs though the oxidation potentials of them were shifted to more positive potentials. Further, the detection limit of 200 nM DOPAC could be achieved at 2.6 nm AuNPs immobilized electrode whereas 500 nM was achieved at 60 nm AuNPs.

As nearly all Mo- and W-dependent enzymes catalyze oxidation and reduction reactions, electrochemistry is an ideal physical method to be applied in the study of the reactivity of these systems. Electrochemical communication between the enzyme and an electrode is of paramount importance and many experimental approaches can be taken to achieve this goal. In this chapter a comprehensive review of the literature of Mo and W enzyme electrochemistry is presented. Direct electrochemistry, where the enzyme is oxidized and reduced at the electrode without any mediators, is experimentally difficult but, when successful, has brought new insight to enzyme function. The use of electron transfer relays (mediators) to achieve better communication with the enzyme is more common and these cases have also been included herein. The application of Mo and W enzyme electrochemistry in biosensors and also in larger scale biotransformations is covered.

Electrochemical biosensors convert biological events to an electrical current. To date most electrochemical biosensors exploit activities of naturally occurring enzymes. Here we demonstrated that insertion of a calmodulin domain into the redox enzyme PQQ-glucose dehydrogenase resulted in a selective Ca(2+) biosensor that could be used to rapidly measure Ca(2+) concentrations in human biological fluids. We were able to convert a point-of-care glucometer into Ca(2+) monitor by refurbishing it with the developed biosensor. We propose that similar engineering strategies may be used to create highly specific electrochemical biosensors to other analytes. Compatibility with cheap and ubiquitous amperometric detectors is expected to accelerate progression of these biosensors into clinical applications.

This article reports the determination of uric acid (UA) in the presence of ascorbic acid (AA) using a self-assembled submonolayer of heteroaromatic dithiol, 2,5-dimercapto-1,3,4-thiadiazole (DMcT), on gold (Au) electrode. Submonolayer to multilayers of DMcT can be prepared on Au electrode by varying the soaking time of Au electrode in 1 mM aqueous solution of DMcT. The formation of submonolayer, monolayer, and multilayers of DMcT on Au electrode was confirmed from its reductive desorption measurements and electrochemical blocking behavior toward ferricyanide. Interestingly, submonolayer of DMcT separates the voltammetric signal of UA from AA by 210 mV, whereas monolayer and multilayers of DMcT fail to separate them. The voltammetric signals of AA and UA are highly stable and reproducible at submonolayer of DMcT. Fast electron transfer, weak hydrogen bonding interactions with AA and UA, and prevention of fouling effect caused by oxidized product of AA can be achieved at submonolayer of DMcT, and thus it successfully separates the voltammetric signals of AA and UA. The practical application of the current system is demonstrated by measuring the concentration of UA in human urine samples without any treatment.

Dimercaptothiadiazole compound, 2,5-dimercapto-1,3,4-thiadiazole (DMcT) forms ‘thin’ monolayers on Au electrode when it was adsorbed from methanol, ethanol or DMSO solutions while it forms ‘thick’ layers on Au electrode from an aqueous solution under identical experimental conditions. Thick DMcT layers formed from aqueous solution effectively blocks the redox reaction of Fe(CN)64-/3- couple in contrast to thin DMcT monolayers. The monolayer thickness did not vary when structurally related DMcT compounds, 5-methyl-1,3,4-thiadiazole-2-thiol or 5-amino-1,3,4-thiadiazole-2-thiol was adsorbed from aqueous and non-aqueous solutions. This indicates that the presence of two thiol groups in DMcT plays a crucial role in the formation of thick and thin DMcT layers on Au electrode when it was adsorbed from aqueous and non-aqueous solutions. Methanol, ethanol, or DMSO solution of DMcT is considered as strong acid because these solvents are able to deprotonate DMcT into DMcT− and thus thin monolayers formed on Au electrode. The deprotonating ability of these solvents was further verified from the observed absorption spectrum characteristic of DMcT− species. On the other hand, an aqueous solution of DMcT is less acidic due to weak deprotonation of DMcT by water and thus DMcT forms thick layer on Au electrode. Interestingly, thin DMcT monolayers formed from non-aqueous solvents separates the voltammetric signals of uric acid and ascorbic acid while thick DMcT layers formed from aqueous solution fails to separate them. Keywords: Self-assembled monolayers, Solvents, Electrochemistry, 2,5-Dimercapto-1,3,4-thiadiazole, Uric acid, Ascorbic acid

We report the electrocatalytic activity of ethylbenzene dehydrogenase (EBDH) from the β-proteobacterium Aromatoleum aromaticum. EBDH is a complex 155 kDa heterotrimeric molybdenum/iron–sulfur/heme protein which catalyzes the enantioselective hydroxylation of nonactivated ethylbenzene to (S)-1-phenylethanol without molecular oxygen as cosubstrate. Furthermore, it oxidizes a wide range of other alkyl-substituted aromatic and heterocyclic compounds to their secondary alcohols. Hydroxymethylferrocenium (FM) is used as an artificial electron acceptor for EBDH in an electrochemically driven catalytic system. Electrocatalytic activity of EBDH is demonstrated with both its native substrate ethylbenzene and the related substrate p-ethylphenol. The catalytic system has been modeled by electrochemical simulation across a range of sweep rates and concentrations of each substrate, which provides new insights into the kinetics of the EBDH catalytic mechanism.

We describe the catalytic voltammograms of the periplasmic arsenite oxidase (Aio) from the chemolithoautotrophic bacterium Rhizobium sp. str. NT-26 that oxidizes arsenite to arsenate. Electrochemistry of the enzyme was accomplished using its native electron transfer partner, cytochrome c552 (cyt c552), as a mediator. The protein cyt c552 adsorbed on a mercaptoundecanoic acid (MUA) modified Au electrode exhibited a stable, reversible one-electron voltammetric response at +275mV vs NHE (pH6). In the presence of arsenite and Aio the voltammetry of cyt c552 is transformed from a transient response to an amplified sigmoidal (steady state) wave consistent with an electro-catalytic system. Digital simulation was performed using a single set of parameters for all catalytic voltammetries obtained at different sweep rates and various substrate concentrations. The obtained kinetic constants from digital simulation provide new insight into the kinetics of the NT-26 Aio catalytic mechanism.

This paper demonstrated the selective determination of folic acid (FA) in the presence of important physiological interferents, ascorbic acid (AA) and uric acid (UA) at physiological pH using electropolymerized film of 5-amino-2-mercapto-1,3,4-thiadiazole (p-AMT) modified glassy carbon (GC) electrode. Bare GC electrode fails to determine the concentration of FA in the presence of AA and UA due to the surface fouling caused by the oxidized products of AA and FA. However, the p-AMT film modified electrode not only separates the voltammetric signals of AA, UA and FA with potential differences of 170 and 410 mV between AA-UA and UA-FA, respectively but also shows higher oxidation current for these analytes. The p-AMT film modified electrode displays an excellent selectivity towards the determination of FA even in the presence of 200-fold AA and 100-fold UA. Using amperometric method, we achieved the lowest detection of 75 nM UA and 100 nM each AA and FA. The amperometric current response was increased linearly with increasing FA concentration in the range of 1.0 x 10(-7) -8.0 x 10(-4) M and the detection limit was found to be 2.3 x 10(-10) M (S/N = 3). The practical application of the present modified electrode was successfully demonstrated by determining the concentration of FA in human blood serum samples. (C) 2009 Elsevier B.V. All rights reserved.

The present study reports the simultaneous determination of ascorbic acid (AA), dopamine (DA) and uric acid (UA) in 0.20 M phosphate buffer solution (pH 5.0) using electropolymerized ultrathin film of 5-amino-2-mercapto-1,3,4-thiadiazole (AMT) on glassy carbon (GC) electrode. The bare GC electrode does not separate the voltammetric signals of AA, DA and UA. However, electropolymerized AMT (p-AMT) modified GC electrode not only resolved the voltammetric signals of AA, DA and UA but also dramatically enhanced their oxidation peak currents when compared to bare GC electrode. The enhanced oxidation currents for AA, DA and UA at p-AMT modified electrode are due to the electrostatic interactions between them and the polymer film. Using amperometric method, we achieved the lowest detection of 75 nM AA, 40 nM DA and 60 nM UA at p-AMT modified electrode. The amperometric current was linearly increased from 200 nM to 0.80 mM for each AA, DA and UA and the lowest detection limit was found to be 0.92, 0.07 and 0.57 nM, respectively (S/N=3). The practical application of the modified electrode was demonstrated by the determination of DA in dopamine hydrochloride injection. (C) 2009 Elsevier B.V. All rights reserved.